The present invention relates to methods of improving the performance of drugs which are metabolized by p450 enzymes, by antisense inhibition of the particular enzyme. Typically, the p450 enzyme is induced by an exogenous substance or by the drug itself.
When a drug is introduced to a biological system, multiple pharmacokinetic processes begin to affect the ultimate efficiency of the drug, determining how rapidly, in what concentration, and for how long the drug will be available to the target organ. In general, lipophilic xenobiotics are metabolized to more polar and hence more readily excretable products. The role metabolism plays in the inactivation of lipid soluble drugs can be quite dramatic. For example, lipophilic barbiturates such as thiopental and phenobarbital would have extremely long half-lives were it not for their metabolic conversion to more water soluble compounds. Many potential anticancer drugs are deemed unbeneficial because their half-life is too brief to achieve any useful therapeutic effect.
The metabolic conversion of an ingested compound (such as a drug or a food additive) into a form which is readily cleared from the body is termed biotransformation or detoxification. Compounds ingested by organisms are generally biotransformed in two phases.
In Phase 1, termed functionalization, a reactive site, such as an amine, thiol, or hydroxyl group, is introduced, generally via an oxidation reaction. In Phase 2, termed conjugation, a water-soluble group is added to the reactive site. Phase 2 typically involves addition of a glucuronic acid, sulfuric acid, acetic acid or amino acid to the compound.
Phase 1 reactions are frequently catalyzed by the cytochrome p450 superfamily of enzymes. In a typical Phase 1 reaction, a cytochrome p450 enzyme uses oxygen and NADH to add a reactive group, such as a hydroxyl radical, to a drug. The reactive intermediates produced may be much more toxic than the parent molecule, and may cause damage to proteins, RNA, and DNA within the cell (Vermeulen, N.P.E., xe2x80x9cRole of metabolism in chemical toxicity,xe2x80x9d in: Ioannides, C., ed., CYTOCHROME P450: METABOLIC AND TOXICOLOGICAL ASPECTS. Boca Raton, Fla.: CRC Press, Inc; 1996, pp 29-53).
Phase 2 conjugation reactions, which generally follow Phase 1 activation reactions, often reduce the toxicity of reactive intermediates formed by Phase 1 reactions. Phase 2 conjugation transforms the drug into a water-soluble compound that can be excreted, e.g. through urine or bile. Several types of conjugation reactions occur in the body, including glucuronidation, sulfation, and glutathione and amino acid conjugation. In some instances, the parent drug may already possess a functional group that forms a conjugate directly. For example, the hydrazide moiety of isoniazide is known to form an acetyl conjugate in a Phase 2 reaction. This conjugate is then a substrate for a Phase 1 type reaction, namely, hydrolysis to isonicotinic acid. Thus, Phase 2 reactions may in some instances actually precede Phase 1 reactions.
Correlations have been noted between altered Phase 1 and/or Phase 2 metabolic activities and increased risk of diseases such as cancers and liver disease, and in adverse drug responses. For example, some drugs (such as acetaminophen) are metabolically converted to reactive intermediates that are toxic to various organs. These toxic reactions may not be apparent at low drug dosages, when subsequent steps or alternative pathways are not overwhelmed or compromised and the availability of endogenous co-substrates (glutathione, glucuronic acid, sulfate) is not limited. When these resources are exhausted, however, the toxic pathway may prevail, resulting in overt organ toxicity or carcinogenesis.
Many drugs and other xenobiotic agents are capable of inducing genes which encode drug-metabolic enzymes, enhancing the levels of these enzymes and, consequently, accelerating the metabolic reactions catalyzed by these enzymes. Such accelerated metabolism may cause a decrease in the half-life and pharmacologic efficacy of the substrate drug. Induction genes encoding drug-metabolizing compounds could exacerbate drug-mediated tissue toxicity by increasing steady-state levels of reactive or toxic intermediates.
A need exists in the art for modulating the pharmacokinetics of various drugs in patients. The present invention achieves this by decreasing the production of one or more specific drug-metabolizing enzymes which are induced either by the drug itself or by another xenobiotic agent to which the patients have been exposed. Decreased drug metabolism results in an increased drug half-life. The dosage of the drug can then be reduced, since the lower dose has equivalent bioavailability to that of a higher dose in the absence of such modulation, and toxicities associated with high drug dosage can be circumvented. Reducing the availability of metabolically toxic pathways thus increases the safety of the drug.
In one aspect, the invention provides a method of improving the pharmacokinetics of a drug administered to a subject, where the drug is known to be metabolized in vivo by a cytochrome p450 enzyme that reduces the effectiveness of the drug. In accordance with the method, one co-administers with the drug a morpholino antisense oligomer effective to reduce synthesis of the drug-metabolizing cytochrome p450 enzyme, by hybridizing to a target RNA molecule which encodes the enzyme. In preferred embodiments of the method, the drug itself induces the drug-metabolizing p450 enzyme, or the subject has been exposed to a xenobiotic agent which induces such an enzyme.
In one embodiment, the antisense oligomer hybridizes to a region of the target RNA molecule which includes the AUG translation start site. In another embodiment, the target RNA molecule is pre-mRNA, and the antisense oligomer hybridizes to a region of the pre-mRNA which includes an intron-exon boundary or an exon-intron boundary.
Preferably, the antisense oligomer is at least 15 nucleotides in length. Preferred oligomers are morpholino oligomers having an uncharged backbone comprising phosphoramidate or, preferably, phosphorodiamidate linkages. The antisense oligomer preferably hybridizes to a region of the target RNA with a Tm greater than 37xc2x0 C. The sequence of the oligonucleotide can be one selected from the group consisting of SEQ ID NOs: 16-35 and 46-47, preferably from SEQ ID NOs: 26-35 and 46-47 (targeted to human RNA sequences), and more preferably from SEQ ID NOs: 27, 30, 34, 35, and 46-47.
The targeted cytochrome p450 enzyme is preferably selected from the group consisting of CYP1A1, CYP1A2, CYP2A6, CYP2B1, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A2, CYP3A4, and CYP6A1 enzymes. In a preferred embodiment, where the subject is a human subject, the cytochrome p450 is preferably selected from the group consisting of CYP1A1, CYP1A2, CYP2A6, CYP2B1, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 enzymes, and more preferably from the group consisting of CYP1A2, CYP2B1, CYP2E1, and CYP3A4 enzymes.
In selected embodiments, the enzyme is CYP2E1, and the drug is acetaminophen, or the enzyme is from the CYP2B or CYP3A subfamily, preferably CYP2B1, and the drug is phenobarbital or hexobarbital. In further embodiments, the enzyme is CYP3A4, and the drug is an antibiotic selected from the group consisting of clarithromycin, erythromycin, rifampicin, rifampin, rifabutin, and rapamycin; or the enzyme is CYP3A4 or CYP1A2, and the drug contains an estrogen or estradiol. In still further embodiments, the enzyme is CYP3A4, the drug is a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor, and the inducing xenobiotic is a CYP3A4-inducing non-nucleoside reverse transcriptase inhibitor.
In a preferred embodiment, the antisense oligomer is administered orally to the subject, typically in an amount of at least 1 mg/kg body weight. In another preferred embodiment, the oligomer is administered transdermally.